Apoptosis and necrosis are two fundamental cell death pathways that may be the end result of response to physiologic activators, physical trauma, or environmental toxins and chemicals. These modes of cell death are associated with a wide range of human pathological conditions, including systemic autoimmunity, cancer, AIDS, and neurodegenerative disorders. Emerging evidence indicates that activation of cysteine proteases of the ICE/CED-3 family is a central mechanism in the execution of apoptosis. Although the mechanisms driving cell necrosis are still obscure, recent studies also point to activation of proteases as a key event in this cell death process. However, the nature of these proteases and their substrates remains largely unknown. Using human antinuclear autoantibodies as probes, we have demonstrated that both apoptosis and necrosis involve the selective, but distinctively different, proteolytic cleavage of a specific set of nuclear protein autoantigens. It is proposed that different proteolytic mechanisms underlie the execution of apoptosis and necrosis, and that dissecting these mechanisms should be helpful for establishing a clear distinction between these cell death processes. The broad, long term objectives of this research is to dissect and differentiate events associated with these mechanisms. Three specific aims are proposed to test our hypothesis. (1) To use a panel of antinuclear autoantibodies where the antigen targets are well characterized nuclear proteins, as probes to define and differentiate patterns of substrate proteolysis associated with Fas-mediated apoptosis and with necrosis induced by the toxic xenobiotic mercury. (2) To characterize proteolytic activities involved in the cleavage of specific autoantigens in both cell death processes. Cell free systems of apoptosis and necrosis will be used in these studies to analyze the inhibition profile of autoantigen cleavage and to determine whether activation of ICE/CED-3 proteases occurs during necrosis. The ability of purified proteases to cleave specific antigens and the identify of specific cleavage sites will be also investigated. (3) To exploit the differences in autoantigen cleavage observed in apoptosis and necrosis for the development of monoclonal antibodies which react specifically with epitopes associated with either apoptotic or necrotic cell death. It is anticipated that these studies should contribute to enhancing our understanding of cell death in molecular terms and establishing additional criteria that would help to differentiate between apoptosis and necrosis.